Chemo-enzymatic synthesis of allyl penta-N-acetyl-chitopentaose

Article abstract of DOI:10.1016/j.bmcl.2005.12.055

Cell density cultivation of recombinant Escherichia coli strains harboring the nodC gene (encoding chitooligosaccharide synthase) from Azorhizobium caulinodans has been previously described as a practical method for the preparation of gram-scale quantities of penta-N-acetyl-chitopentaose. We have now extended this method to the production of allylated derivative of penta-N-acetyl-chitopentaose by using allyl 2-acetamido-2-deoxy-β-d- glucopyranoside (2) as the initial acceptor for the synthesis of target pentaoside in vivo.

Full text of DOI:10.1016/j.bmcl.2005.12.055

Bioorganic & Medicinal Chemistry Letters 16 (2006) 2042–2043
Chemo-enzymatic synthesis of allyl penta-N-acetyl-chitopentaose
Gang-Liang Huang, Da-Wei Zhang, Hong-Juan Zhao,
Hou-Cheng Zhang and Peng-George Wang*
School of Life Science, Shandong University, Jinan City 250100, China
Received 26 September 2005; revised 3 December 2005; accepted 14 December 2005
Available online 5 January 2006
Abstract—Cell density cultivation of recombinant Escherichia coli strains harboring the nodC gene (encoding chitooligosaccharide
synthase) from Azorhizobium caulinodans has been previously described as a practical method for the preparation of gram-scale
quantities of penta-N-acetyl-chitopentaose. We have now extended this method to the production of allylated derivative of
penta-N-acetyl-chitopentaose by using allyl 2-acetamido-2-deoxy-b-D-glucopyranoside (2) as the initial acceptor for the synthesis
of target pentaoside in vivo.
Ó 2005 Elsevier Ltd. All rights reserved.
It has already been shown that Escherichia coli cells
overproducing the recombinant proteins NodC (i.e.,
N-acetylglucosaminyltransferase) accumulated high
intracellular concentrations of penta-N-acetyl-chitopen-
taose.1 It has furthermore recently been proved that the
synthesis of chitin oligosaccharides by NodC proceeds
by the addition of GlcNAc residues from the donor
UDP-GlcNAc to O-4 of the nonreducing-terminal resi-
due of the growing chain.2 The enzyme is able to use free
GlcNAc as acceptor. The fact that allyl 2-acetamido-2-
deoxy-b-D-glucopyranoside (2) as starting sugar can be
used with recombinant E. coli strains expressing NodC
gene in vitro has already been described.3 We herein de-
scribe the synthesis of allyl penta-N-acetyl-chitopenta-
ose by using 2 as acceptor and a batch culture of
E. coli strain BL21 containing a plasmid carrying the
cloned nodC gene from Azorhizobium caulinodans.
OH
O
OH
O
HO
HO
HO
HO
NaH, CH2=CHCH2Br
DMF, LiBr, 82%
OH
OCH2CH=CH2
AcHN
AcHN
2
1
OH
O
chitooligosaccharide synthase
(nodC gene encode)
HO
HO
OCH2CH=CH2
OH
, 65%
UDP-GlcNAc UDP
4
AcHN
2
OH
O
OH
HO
HO
O
O
O
O
OCH2CH=CH2
HO
HO
AcHN
3
AcHN
AcHN
3
Scheme 1. The synthetic route of 3.
can notably improve the stereoselectivity of the glycosyl-
ation of 1. This one-step glycoside synthesis compares
well with the standard peracetylation, selective anomeric
deprotection, O-alkylation at C1, and deacetylation
steps.5
2-Acetamido-2-deoxy-D-glucose 1 and LiBr (2 equiv)
were suspended in DMF (1 mL per mmol of 1) and suc-
cessively treated with sodium hydride (1.3 equiv) and al-
lyl bromide (3 equiv) at room temperature for 3 h. The
crude product was directly purified by flash chromatog-
raphy (4:1, EtOAc/MeOH), and the b-allyl glycoside 24
was obtained in 82% yield (Scheme 1). Addition of LiBr
Cell density cultures were carried out as previously de-
scribed1 in 2 or 10 L bioreactors containing an initial cul-
ture volume of 1 or 7 L, respectively. Compound 2 was
added to culture system. The E. coli strain BL21(DE3)
containing a plasmid carrying the cloned nodC gene from
A. caulinodans strain E1R6 was used as the source of
NodC protein. The culture time lasted 48 h.
Keywords: Allyl 2-acetamido-2-deoxy-b-D-glucopyranoside; Penta-N-
acetyl-chitopentaose; Chemo-enzymatic synthesis.
After centrifugation of the culture broth, chitooligosac-
charide was recovered exclusively in the pellet containing
*
Corresponding author. Tel.: +86 531 8836 6078; e-mail: hgl226@
126.com
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.12.055

G.-L. Huang et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2042–2043
2043
the bacterial cells. After disruption of the cells by boiling,
Supplementary material
cell debris was removed by centrifugation and the chitool-
igosaccharide was purified by activated charcoal adsorp-
tion and aq ethanol (55%, v/v) elution. The crude product
was further purified by size exclusion chromatography on
Biogel P2. The NMR and MS data confirmed the identi-
fication of the obtained sample as target pentaoside 3 (for
details, see Supporting information). The yield was 65%.
Therefore, 2 containing the hydrophobic allyl group
linked to C-1 is acceptor for NodC in vivo. The most obvi-
ous explanation for this phenomenon is that the allylated
derivative 2 of GlcNAc does not influence the binding
affinity of NodC for the oligosaccharide intermediate
and therefore leads to an elongation with additional Glc-
NAc units.
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/
j.bmcl.2005.12.055.
References and notes
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As seen from the above-mentioned synthetic route, it
demonstrates the possibility of using recombinant
technology for the production of high-value substituted
oligosaccharides in vivo by using the derivatives of
monosaccharides as the initial acceptors that are
involved in the introduction of the substitutions.
6. Kamst, E.; Pilling, J.; Raamsdonk, L. M.; Lugtenberg, B. J.
J.; Spaink, H. P. J. Bacteriol. 1997, 179, 2103.